The genome sequence of the Mournful Wasp, Pemphredon lugubris (Fabricius, 1793)

We present a genome assembly from an individual male Pemphredon lugubris (the Mournful Wasp; Arthropoda; Insecta; Hymenoptera; Crabronidae). The genome sequence is 328.1 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.88 kilobases in length. Gene annotation of this assembly on Ensembl identified 10,335 protein coding genes.

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The final assembly has a total length of 328.1 Mb in 1358 sequence scaffolds with a scaffold N50 of 64.8 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (95.65%) of the assembly sequence was assigned to 5 chromosomal-level scaffolds, representing 5 autosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The assembly is haploid.The order and orientation of scaffolds are uncertain on chromosome 1 in the region 31.55 to 42.17 Mb, and chromosome 2 in the region 17.43 to 65.55 Mb.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Background
The Mournful Wasp, Pemphredon lugubris, is a dark, mediumsized (7.5-12 mm) solitary wasp in the family Pemphredonidae.It occurs across Europe and North America and is common and widespread in the UK.It is the largest and the most frequently recorded Pemphredon species in the UK and the only one from the subgenus Pemphredon present.Other subgenera of Pemphredon are currently in a state of taxonomic flux, although Pemphredon Pemphredon remains relatively stable.The integument is entirely black in colour, from which the common name is derived, and it has a petiolate abdomen.The forewing has two submarginal cells, with vein 2m-cu meeting the second submarginal, differentiating it from all other Pemphredon species in the UK except P. morio.It can be distinguished from this species by the lack of a tooth beneath the base of the antennae and a non-emarginate clypeus.
It occurs across a variety of habitats and may be found anywhere there is suitable deadwood.Nests are constructed in cavities in dead and decaying wood and are typically formed of a main tunnel with several subsidiary tunnels branching off, each with up to several cells (Bitsch et al., 2001).Females hunt aphids, provisioning each cell with up to 40 (Lomholdt, 1975).Other Hemipteran prey may be occasionally used (Whitehead, 1990), but this remains uncertain due to the high prevalence of nest usurpation by Pemphredon.It is a univoltine species with a flight period from May to September.It is attacked by the kleptoparasites Pseudomalus violaceus and auratus (Blösch, 2000), which gain access to the Pemphredon nests by laying eggs on aphids which are subsequently taken by Pemphredon (Bitsch, 2022).
The complete genome sequence for this species will facilitate studies into the evolution of hunting strategies, sociality, reproductive systems and Hymenopteran taxonomy.

Genome sequence report
The genome was sequenced from one male Pemphredon lugubris (Figure 1) collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (51.77,.A total of 58-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 82-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 421 missing joins or mis-joins, reducing the assembly length by 0.03% and the scaffold number by 23.58%, and increasing the scaffold N50 by 118.68%.( Sheerin et al., 2023).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.
Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from abdomen tissue of iyPemLugu1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from head tissue of iyPemLugu1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012).
The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Pemphredon lugubris assembly (GCA_933228935.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Major concerns
The work in general is very good and the method used for sequencing quite robust.The manuscript could be a bit more descriptive about some aspects, but I understand that it is more a report than a research paper.The main weakness of the study is that the annotation was completed using the Uniprot database without transcriptomes supporting the gene annotation.I believe that the quality of the genome assembly is very good but in terms of gene annotation should be considered a draft genome to be improved in the future.

Minors
Title: I recommend adding the insect' order and family to the title.
○ "P.morio": genera cannot be abbreviated if the complete species name was not mentioned before.

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Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Pemphredon lugubris, iyPemLugu1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 328,159,117 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (74,671,469 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (64,765,430 and 48,191,648 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKOGF01/dataset/CAKOGF01/snail.

Figure 3 .
Figure 3. Genome assembly of Pemphredon lugubris, iyPemLugu1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKOGF01/dataset/CAKOGF01/blob.

Figure 4 .
Figure 4. Genome assembly of Pemphredon lugubris, iyPemLugu1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAKOGF01/dataset/CAKOGF01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Pemphredon lugubris, iyPemLugu1.1:Hi-C contact map of the iyPemLugu1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=bxzUvAXrQlGeHrz_iSh0VQ.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes
Reviewer Expertise: Non-model population genomicsI confirm that I

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.